Substrate profiling of PRMT1 reveals amino acid sequences that extend beyond the "RGG" paradigm

Protein arginine methyltransferase 1 (PRMT1) catalyzes the mono- and dimethylation of certain protein arginine residues. Although this posttranslational modification has been implicated in many physiological processes, the molecular basis for PRMT1 substrate recognition is poorly understood. Most modified arginine residues in known PRMT1 substrates reside in repeating "RGG" sequences. However, PRMT1 also specifically methylates Arg3 of histone H4 in a region that is not glycine-arginine rich, suggesting that PRMT1 substrates are not limited to proteins bearing "RGG" sequences. Because a systematic evaluation of PRMT1 substrate specificity has not been performed, it is unclear if the "RGG" sequence accurately represents the consensus target for PRMT1. Using a focused peptide library based on a sequence derived from the in vivo substrate fibrillarin we observed that PRMT1 methylated substrates that had amino acid residues other than glycine in the "RX (1)" and "RX (1)X (2)" positions. Importantly, eleven additional PRMT1 substrate sequences were identified. Our results also illustrate that the two residues on the N-terminal side of the modification site are important and need not both be glycine. PRMT1 methylated the eukaryotic initiation factor 4A1 (eIF4A1) protein, which has a single "RGG" sequence. Methylation of eIF4A1 and the similar eIF4A3 could be affected using single site mutations adjacent to the modification site, demonstrating the importance of amino acid sequence in PRMT1 protein substrates. Dimethylation of the parent library peptide was shown to occur through a dissociative mechanism. In summary, PRMT1 selectively recognizes a set of amino acid sequences in substrates that extend beyond the "RGG" paradigm.     

PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex

Protein arginine methyltransferase 3 (PRMT3) comprises a region not required for catalytic activity in its amino-terminus and the core domain catalyzing protein arginine methylation. PRMT3 has been shown to interact with the 40S ribosomal protein S2 (rpS2) and methylate arginine residues in the arginine-glycine (RG) repeat region in the amino-terminus of rpS2. We investigated the biological implications of this interaction by delineating the domains that mediate binding between PRMT3 and rpS2. The rpS2 (100-293 amino acids) domain, but not the amino-terminus of rpS2 that includes the RG repeat region was essential for binding to PRMT3 and was susceptible to degradation. The amino-terminus of PRMT3, but not its catalytic core was required for binding to and the stability of rpS2. Overexpressed rpS2 was ubiquitinated in cells, but expression of PRMT3 reduced this ubiquitination and stabilized the rpS2 protein. Recombinant PRMT3 formed an active enzyme complex with endogenous rpS2 in vitro. Recombinant rpS2 in molar excess modestly increased the enzymatic activity of PRMT3 in vitro. Our results suggest that in addition to its catalytic function, PRMT3 may control the level of rpS2 protein in cells by inhibiting ubiquitin-mediated proteolysis of rpS2, while rpS2 may regulate the enzymatic activity of PRMT3 as a likely non-catalytic subunit.     

Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.     

PRMT3 is a ribosomal protein methyltransferase that affects the cellular levels of ribosomal subunits

The mammalian protein arginine methyltransferase 3 (PRMT3) catalyzes the formation of asymmetric (type I) dimethylarginine in vitro. As yet, natural substrates and cellular pathways modulated by PRMT3 remain unknown. Here, we have identified an ortholog of PRMT3 in fission yeast. Tandem affinity purification of fission yeast PRMT3 coupled with mass spectrometric protein identification revealed that PRMT3 associates with components of the translational machinery. We identified the 40S ribosomal protein S2 as the first physiological substrate of PRMT3. In addition, a fraction of yeast and human PRMT3 cosedimented with free 40S ribosomal subunits, as determined by sucrose gradient velocity centrifugation. The activity of PRMT3 is not essential since prmt3-disrupted cells are viable. Interestingly, cells lacking PRMT3 showed an accumulation of free 60S ribosomal subunits resulting in an imbalance in the 40S:60S free subunits ratio; yet pre-rRNA processing appeared to occur normally. Our results identify PRMT3 as the first type I ribosomal protein arginine methyltransferase and suggest that it regulates ribosome biosynthesis at a stage beyond pre-rRNA processing.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

PRMT3 is a distinct member of the protein arginine N-methyltransferase family. Conferral of substrate specificity by a zinc-finger domain

S-Adenosyl-l-methionine-dependent protein arginine N-methyltransferases (PRMTs) catalyze the methylation of arginine residues within a variety of proteins. At least four distinct mammalian family members have now been described, including PRMT1, PRMT3, CARM1/PRMT4, and JBP1/PRMT5. To more fully define the physiological role of PRMT3, we characterized its unique putative zinc-finger domain and how it can affect its enzymatic activity. Here we show that PRMT3 does contain a single zinc-finger domain in its amino terminus. Although the zinc-liganded form of this domain is not required for methylation of an artificial substrate such as the glutathione S-transferase-fibrillarin amino-terminal fusion protein (GST-GAR), it is required for the enzyme to recognize RNA-associated substrates in RAT1 cell extracts. The recombinant form of PRMT3 is inhibited by high concentrations of ZnCl(2) as well as N-ethylmaleimide, reagents that can modify cysteine sulfhydryl groups. We found that we could distinguish PRMT family members by their sensitivity to these reagents; JBP1/PRMT5 and Hsl7 methyltransferases were inhibited in a similar manner as PRMT3, whereas Rmt1, PRMT1, and CARM1/PRMT4 were not affected. We were also able to define differences in these enzymes by their sensitivity to inhibition by Tris and free arginine. Finally, we found that the treatment of RAT1 cell extracts with N-ethylmaleimide leads to a loss of the major PRMT1-associated activity that was immune to inhibition under the same conditions as a GST fusion protein. These results suggest that native forms of PRMTs can have different properties than their GST-catalytic chain fusion protein counterparts, which may lack associated noncatalytic subunits.     

Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming ω-NG-monomethylated arginine residues

Full-length human protein arginine methyltransferase 7 (PRMT7) expressed as a fusion protein in Escherichia coli was initially found to generate only ω-N(G)-monomethylated arginine residues in small peptides, suggesting that it is a type III enzyme. A later study, however, characterized fusion proteins of PRMT7 expressed in bacterial and mammalian cells as a type II/type I enzyme, capable of producing symmetrically dimethylated arginine (type II activity) as well as small amounts of asymmetric dimethylarginine (type I activity). We have sought to clarify the enzymatic activity of human PRMT7. We analyzed the in vitro methylation products of a glutathione S-transferase (GST)-PRMT7 fusion protein with robust activity using a variety of arginine-containing synthetic peptides and protein substrates, including a GST fusion with the N-terminal domain of fibrillarin (GST-GAR), myelin basic protein, and recombinant human histones H2A, H2B, H3, and H4. Regardless of the methylation reaction conditions (incubation time, reaction volume, and substrate concentration), we found that PRMT7 only produces ω-N(G)-monomethylarginine with these substrates. In control experiments, we showed that mammalian GST-PRMT1 and Myc-PRMT5 were, unlike PRMT7, able to dimethylate both peptide P-SmD3 and SmB/D3 to give the expected asymmetric and symmetric products, respectively. These experiments show that PRMT7 is indeed a type III human methyltransferase capable of forming only ω-N(G)-monomethylarginine, not asymmetric ω-N(G),N(G)-dimethylarginine or symmetric ω-N(G),N(G')-dimethylarginine, under the conditions tested.     

Site specificity of histone H4 methylation by wheat germ protein-arginine N-methyltransferase

CNBr treatment of calf thymus [methyl-14C]histone H4, methylated in vitro with S-adenosyl-L-[methyl-14C]methionine by a highly histone-specific wheat germ protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), produced two peptide fragments corresponding to residues 1-83 and 84-102, with the former being radioactive. Two-dimensional peptide mapping of the chymotryptic and tryptic digest of [methyl-14C]histone H4 and analysis of the chymotryptic digest on HPLC have shown that only a single peptide is radiolabeled. In order to define the exact site of methylation (arginine residue), the radioactive peptide from the chymotryptic digest of [methyl-14C]histone H4 was further purified on HPLC by linear and then isocratic elution. The purified chymotryptic peptide was then digested with trypsin and purified on HPLC, and its amino acid composition was determined on HPLC. These results indicate that the peptide corresponding to residues 24-35 of histone H4 is radiolabeled. Since this peptide contains a single arginine residue at position 35, we have concluded that the enzyme is specific not only to the protein substrate but also to the methylation site.     

Kinetic mechanism of protein arginine methyltransferase 1

Protein arginine methyltransferases (PRMTs) are SAM-dependent enzymes that catalyze the mono- and dimethylation of peptidyl arginine residues. Although all PRMTs produce monomethyl arginine (MMA), type 1 PRMTs go on to form asymmetrically dimethylated arginine (ADMA), while type 2 enzymes form symmetrically dimethylated arginine (SDMA). PRMT1 is the major type 1 PRMT in vivo, thus it is the primary producer of the competitive NOS inhibitor, ADMA. Hence, potent inhibitors, which are highly selective for this particular isozyme, could serve as excellent therapeutics for heart disease. However, the design of such inhibitors is impeded by a lack of information regarding this enzyme's kinetic and catalytic mechanisms. Herein we report an analysis of the kinetic mechanism of human PRMT1 using both an unmethylated and a monomethylated substrate peptide based on the N-terminus of histone H4. The results of initial velocity and product and dead-end inhibition experiments indicate that PRMT1 utilizes a rapid equilibrium random mechanism with the formation of dead-end EAP and EBQ complexes. This mechanism is gratifyingly consistent with previous results demonstrating that PRMT1 catalyzes substrate dimethylation in a partially processive manner.     

Peptide backbone conformation affects the substrate preference of protein arginine methyltransferase I

Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.     

A protein arginine N-methyltransferase 1 (PRMT1) and 2 heteromeric interaction increases PRMT1 enzymatic activity

Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene expression by methylating arginine residues within target proteins. PRMT1 is responsible for most cellular arginine methylation activity and can work independently or in collaboration with other PRMTs. In this study, we demonstrate a direct interaction between PRMT1 and PRMT2 using co-immunoprecipitation, bimolecular fluorescence complementation, and enzymatic assays. As a result of this interaction, PRMT2 stimulated PRMT1 activity, affecting its apparent V(max) and K(M) values in vitro and increasing the production of methylarginines in cells. Active site mutations and regional deletions from PRMT1 and -2 were also investigated, which demonstrated that complex formation required full-length, active PRMT1. Although the inhibition of methylation by adenosine dialdehyde prevented the interaction between PRMT1 and -2, it did not prevent the interaction between PRMT1 and a truncation mutant of PRMT2 lacking its Src homology 3 (SH3) domain. This result suggests that the SH3 domain may mediate an interaction between PRMT1 and -2 in a methylation-dependent fashion. On the basis of our findings, we propose that PRMT1 serves as the major methyltransferase in cells by forming higher-order oligomers with itself, PRMT2, and possibly other PRMTs.     

PRMT7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity

We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of omega-NG-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli as a glutathione S-transferase (GST) fusion protein. We found that GST-tagged PRMT7 catalyzes the S-adenosyl-[methyl-3H]-l-methionine-dependent methylation of the synthetic peptide GGPGGRGGPGG-NH2 (R1). The radiolabeled peptide was purified by high-pressure liquid chromatography and acid hydrolyzed to free amino acids. When the hydrolyzed products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species which co-migrated with an omega-NG-monomethylarginine standard. Surprisingly, GST-PRMT7 was not able to catalyze the in vitro methylation of a GST-fibrillarin (amino acids 1-148) fusion protein (GST-GAR), a methyl-accepting substrate for the previously characterized PRMT1, PRMT3, PRMT4, PRMT5, and PRMT6 enzymes. Nor was it able to methylate myelin basic protein or histone H2A, in vitro substrates of PRMT5. This specificity distinguishes PRMT7 from all of the other known arginine methyltransferases. An additional unique feature of PRMT7 is that it seems to have arisen from a gene duplication event and contains two putative AdoMet-binding motifs. To see if both motifs were necessary for activity, each putative domain was expressed as a GST-fusion and tested for activity with peptides R1 and R2 (acetyl-GGRGG-NH2). These truncated proteins were enzymatically inactive, suggesting that both domains are required for functionality.     

Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of omega-N(G)-monomethylarginine in calf thymus histone H2A

The HSL7 (histone synthetic lethal 7) gene in the yeast Saccharomyces cerevisiae encodes a protein with close sequence similarity to the mammalian PRMT5 protein, a member of the class of protein arginine methyltransferases that catalyses the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N'(G)-dimethylarginine residues in a number of methyl-accepting species. A full-length HSL7 construct was expressed as a FLAG-tagged protein in Saccharomyces cerevisiae. We found that FLAG-tagged Hsl7 effectively catalyses the transfer of methyl groups from S-adenosyl-[methyl-3H]-L-methionine to calf thymus histone H2A. When the acid-hydrolysed radiolabelled protein products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species that co-migrated with an omega-N(G)-monomethylarginine standard. No radioactivity was observed that co-migrated with either the asymmetric or symmetric dimethylated derivatives. In control experiments, no methylation of histone H2A was found with two mutant constructs of Hsl7. Surprisingly, FLAG-Hsl7 does not appear to effectively catalyse the in vitro methylation of a GST (glutathione S-transferase)-GAR [glycine- and arginine-rich human fibrillarin-(1-148) peptide] fusion protein or bovine brain myelin basic protein, both good methyl-accepting substrates for the human homologue PRMT5. Additionally, FLAG-Hsl7 demonstrates no activity on purified calf thymus histones H1, H2B, H3 or H4. GST-Rmt1, the GST-fusion protein of the major yeast protein arginine methyltransferase, was also found to methylate calf thymus histone H2A. Although we detected Rmt1-dependent arginine methylation in vivo in purified yeast histones H2A, H2B, H3 and H4, we found no evidence for Hsl7-dependent methylation of endogenous yeast histones. The physiological substrates of the Hsl7 enzyme remain to be identified.     

Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior

The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.     

Discovery of new potent protein arginine methyltransferase 5 (PRMT5) inhibitors by assembly of key pharmacophores from known inhibitors

Protein arginine methyltransferase 5 (PRMT5) is an epigenetics related enzyme that has been validated as a promising therapeutic target for human cancer. Up to now, two small molecule PRMT5 inhibitors has been put into phase I clinical trial. In the present study, a series of candidate molecules were designed by combining key pharmacophores of formerly reported PRMT5 inhibitors. The in vitro PRMT5 inhibitory testing of compound 4b14 revealed an IC₅₀ of 2.71?μM, exhibiting high selectivity over PRMT1 and PRMT4 (>70-fold selective). As expected, 4b14 exhibited potent anti-proliferative activity against a panel of leukemia and lymphoma cells, including MV4-11, Pfeiffer, SU-DHL-4 and KARPAS-422. Besides, 4b14 showed significant cell cycle arrest and apoptosis-inducing effects, as well as reduced the cellular symmetric arginine dimethylation level of SmD3 protein. Finally, affinity profiling analysis indicated that hydrophobic interactions, π-π stacking and cation-π actions made the major contributions to the overall binding affinity. This scaffold provides a new chemical template for further development of better lead compounds targeting PRMT5.     

Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases

The conjugates of an adenosine mimetic and oligo-l -arginine or oligo-d -arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l -methionine (SAM) and S-adenosyl-l -homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.